Bone marrow transplantation (BMT) is used to treat a variety of diseases including hematological and lymphoid malignancies, anemias, autoimmune disorders and others. The critical component of each BMT is hematopoietic stem cells (HSC), which are defined by their ability to self-renew and to differentiate into all blood cell types. The objective of this grant is to understand the role of early T cells in regulating HSC selfrenewal and expansion. Recent studies from the Lodish laboratory showed that coculture of embryonic day 15.5 (E15.5) mouse fetal liver (FL) CD3+ cells with enriched adult bone marrow HSCs led to expansion of HSC numbers and activities. These CD3+ cells specifically express several secreted growth factors including Insulin-like Growth factor 2 (IGF-2) and Angiopoietin-like 2 and 3 (Angptl 2 and 3). Inclusion of these factors in a serum-free culture medium also containing Stem Cell Factor (SCF), Thrombopoietin (TPO), and Fibroblast Growth Factor 1 supported a 24 to 30 fold net expansion of long-term HSC, as observed by reconstitution analysis. In preliminary studies we showed that significant proportion of E15.5 FL CD3+ cells are CD4" and CDS" but positive for either T cell receptor ap or y6 and thus likely are early T cells. In this proposal I will: 1) Characterize phenotype and activation/memory status of subpopulations of CD3+ cells in E15.5 FL, and test the hypothesis that these cells are early natural killer T cells;examine ability of subpopulations of FL cells to support ex vivo self-renewal and expansion of HSCs;examine expression pattern of genes known to promote self-renewal of HSCs in the subpopulations of CD3+ cells that promote ex vivo self-renewal and expansion of HSCs. 2) Examine adult bone marrow, spleen and liver for the presence of subpopulations of CD3+ cells similar to subpopulations of E15.5 FL CD3+ cells that are capable of ex vivo expansion of BM HSCs. 3) Examine gene expression profile of purified FL and adult CD3+ cell populations that support HSC expansion in culture using microarray analysis, to identify potentially novel proteins with HSC supportive function. The clinical practice data suggests that the number of patients that are potential candidates for BMT has increased significantly in the past decade while the availability of compatible donors did not follow this trend. Our capability to expand the number of available HSC, would not only lead to increase in the number of potential donors and improved engraftment, but it would also promote development of new therapeutic approaches using HSC manipulation. (End of Abstract)